This proposal addresses a long-standing problem in biology - the relative contribution of replicative DNA polymerases (Pol) in eukaryotes in guarding genome stability and protecting from diseases stemming from faults in replication. Remarkable progress in the field in recent years has led to the understanding that multiple DNA polymerases are required for accurate replication, recombination and repair of the eukaryotic genome. However, the roles of individual polymerases are far from being understood. We designed an integral approach to study roles of Pols. We will combine in vitro biochemical and in vivo genetic methods. The advantage over the existing methods is the use of the supermutagenic base analog, hydroxylaminopurine (HAP) and strategic DNA polymerase mutants. HAP makes the detection of DNA strand-specific errors possible and is based on years of Dr. Pavlov's research. Mutator strains possessing Pols with reduced base selectivity and disabled proofreading exonuclease will allow us to track individual Pols in vivo. The goal of the study will be pursued in the three specific aims. First is the genetic study of DNA Pol interplay by double mutant analysis. Using mutants with inaccurate Pol a as a reference, we expect to define the order of DNA Pols transactions on the lagging and leading DNA strands. Specific Aim 2 is the determination of error signatures of Pols and their inaccurate variants in vitro in the URA3 gene. Specific Aim 3 is the determination of the relative roles of DNA polymerases in genome stability. In one approach we will study the distribution of mutation hotspots in the reporter placed near the replication origin in strains with inaccurate Pol a. The involvement of inaccurate Pol a in the initiation of replication in vivo will result in a unique periodic distribution of mutations coinciding with the boundaries of Okazaki fragments. For the first time, this will provide an estimate of the size of Okazaki fragments in vivo. In second approach, the mutation spectra produced by Pols in vitro and generated in vivo in the same reporter gene will be compared by powerful statistical approaches. The mutation signatures of the DNA polymerases found in each of the in vivo spectra will reveal the extent of their participation in the replication of the leading and lagging strands. We expect that Aims 1-3 will lead to a better understanding of the Pol arrangement at the replication fork. The research aims the fundamental human health-oriented biological problem of the roles of different DNA polymerases in the replication fork in eukaryotes. The mutations generated by errors of the DNA polymerases are among the primary causes of cancer. The work will result in a database of mutations generated by inaccurate DNA polymerases, which could be used to find the same mutation signature in human cancers. We will also find a combination of polymerase alleles that synergistically destabilize the genome and create a predisposition to cancer and other diseases. PUBLIC HEALTH RELEVANCE: The project seeks to estimate the relative roles of replicative DNA polymerases at replication fork in eukaryotes and the genetic consequences and impact on human health of mutations in DNA polymerases compromising their fidelity.